1. Field of the Invention
The present invention relates to a method of detecting a gene as an amplified product by gene amplification and a reagent kit for the method, more particularly, a method for accurately detecting a gene as an amplified product or a byproduct by reducing the turbidity which is generated in a gene amplification solution and may disturb accurate detection of the gene or the byproduct and a reagent kit for the method.
2. Description of Related Art
A variety of gene amplification methods are effectively utilized in identification of genetic diseases, cancer, microorganisms or the like in medical scenes and others, because of their simplicity and rapidity. Among such methods, PCR (polymerase chain reaction) is often used particularly in the medical field since the method is good in sensitivity (Japanese patent publication HEI 4(1992)-67957). Also, attention is being given to a LAMP (Loop-mediated Isothermal Amplification) method because this method provides a specific reaction and can amplify a large amount of an amplified product (Japanese patent 3313358).
Further, in recent years, a quantitative PCR method has been developed which enables determination of the amount of existing target genes as well as amplification of target genes. According to this method, the degree of the amplification of the target genes can be detected in real time fashion and the amount of the target genes can be determined with respect to the time when amplification is detected.
The real-time detection of gene amplification is roughly classified into two types, i.e., a fluorescent method and a turbidimetric method. Particularly, according to the turbidimetric method, quantitative gene amplification can be performed by detecting amplified products in real time fashion with use of a precipitation as a turbidity index. The precipitation is produced in a solution by reaction of magnesium with pyrophosphoric acid which is generated by gene amplification. Furthermore, a detector used for the turbidimetric method is simple. In these respects, the turbidimetric method is superior.
However, solution for gene amplification typically contains not only enzymes such as polymerase, reverse transcriptase and the like but also surfactants for improving the stability and reactivity of the enzymes. If amplification reaction is carried out with use of such solution with heating to about 65° C., the solution become turbid owing to the surfactants. For this reason, in the medical scene or the like, when the amplification of target genes is detected by the turbidimetric method, the background from the solution increase, and therefore, it is impossible to detect the gene amplification with high sensitivity and accuracy.